Novel Approach for High-Throughput Metabolic Screening of Whole Plants by Stable Isotopes.

Here, we demonstrate whole-plant metabolic profiling by stable isotope labeling and combustion isotope-ratio mass spectrometry for precise quantification of assimilation, translocation, and molecular reallocation of (13)CO2 and (15)NH4NO3 The technology was applied to rice (Oryza sativa) plants at different growth stages. For adult plants, (13)CO2 labeling revealed enhanced carbon assimilation of the flag leaf from flowering to late grain-filling stage, linked to efficient translocation into the panicle. Simultaneous (13)CO2 and (15)NH4NO3 labeling with hydroponically grown seedlings was used to quantify the relative distribution of carbon and nitrogen. Two hours after labeling, assimilated carbon was mainly retained in the shoot (69%), whereas 7% entered the root and 24% was respired. Nitrogen, taken up via the root, was largely translocated into the shoot (85%). Salt-stressed seedlings showed decreased uptake and translocation of nitrogen (69%), whereas carbon metabolism was unaffected. Coupled to a gas chromatograph, labeling analysis provided enrichment of proteinogenic amino acids. This revealed significant protein synthesis in the panicle of adult plants, whereas protein biosynthesis in adult leaves was 8-fold lower than that in seedling shoots. Generally, amino acid enrichment was similar among biosynthetic families and allowed us to infer labeling dynamics of their precursors. On this basis, early and strong (13)C enrichment of Embden-Meyerhof-Parnas pathway and pentose phosphate pathway intermediates indicated high activity of these routes. Applied to mode-of-action analysis of herbicides, the approach showed severe disturbance in the synthesis of branched-chain amino acids upon treatment with imazapyr. The established technology displays a breakthrough for quantitative high-throughput plant metabolic phenotyping.

Mental but no bio-physiological long-term habituation to repeated social stress: A study on soldiers and the influence of mission abroad.

Soldiers regularly participate in missions abroad and subjectively adapt to this situation. However, they have an increased lifetime cardiovascular risk compared to other occupational groups. To test the hypothesis that foreign deployment results in different stress habituation patterns, we investigated long-term psychological and bio-physiological stress responses to a repeated social stress task in healthy soldiers with and without foreign deployment. Ninety-one female and male soldiers from the BEST study (German armed forces deployment and stress) participated three times in the Trier Social Stress Test for groups (TSST-G) prior to, 6-8 weeks after and 1 year after the mission abroad and were compared to a control group without foreign deployment during the study period. They completed the State-Trait-Anxiety Inventory scale (STAI), the Primary Appraisal Secondary Appraisal questionnaire (PASA) and the Multidimensional Mood State Questionnaire (MDBF). Salivary cortisol and α-amylase, blood pressure, heart rate and heart rate variability were determined. Soldiers showed mental habituation over the three times with a significant decrease after the TSST-G in anxiousness (STAI) and cognitive stress appraisal (PASA), they were calmer and reported better mood (MDBF). Prior to the social stress part, the mood (MDBF) declined significantly. None of the biological and physiological markers showed any adaptation to the TSST-G. Mission abroad did not significantly influence any measured psychobiological marker when compared to soldiers without foreign deployment. Foreign deployment does not result in alterations in psychobiological social stress response patterns over 1 year after mission abroad which indicates that adaptation to acute social stress is highly maintained in healthy soldiers. The discrepancy between subjective perception and objective stress response has numerous clinical implications and should receive more attention.

Structural Equation Modeling of a Global Stress Index in Healthy Soldiers.

Accumulation of stress is a prognostic trigger for cardiovascular disease. Classical scores for cardiovascular risk estimation typically do not consider psychosocial stress. The aim of this study was to develop a global stress index (GSI) from healthy participants by combining individual measures of acute and chronic stress from childhood to adult life. One-hundred and ninety-two female and male soldiers completed the Perceived Stress Scale (PSS4), Trier Inventory for Chronic Stress (TICS), Hospital Anxiety and Depression Scale (HADS), Childhood Trauma Questionnaire (CTQ), Posttraumatic Diagnostic Scale Checklist (PDS), and the Deployment Risk and Resilience Inventory (DRRI-2). The underlying structure for the GSI was examined through structural equation modeling. The final hierarchical multilevel model revealed fair fit by taking modification indices into account. The highest order had a g-factor called the GSI. On a second level the latent variables stress, HADS and CTQ were directly loading on the GSI. A third level with the six CTQ subscales was implemented. On the lowest hierarchical level all manifest variables and the DRRI-2/PDS sum scores were located. The presented GSI serves as a valuable and individual stress profile for soldiers and could potentially complement classical cardiovascular risk factors.

Social Stress-Induced Oxidative DNA Damage Is Related to Prospective Cardiovascular Risk.

Psychosocial stress increases cardiovascular risk, which coincides with enhanced oxidative DNA damage. Increased sympathetic tone-related catecholamine release causes oxidative stress, which contributes to catecholamine-related cardiotoxicity. Therefore, we tested the hypothesis whether acute psychosocial stress induces oxidative DNA damage, its degree being related to the cardiovascular risk profile and depending on the sympathetic stress response. After assessment of the prospective cardiovascular Münster score (PROCAM) to determine the risk of acute myocardial infarction, 83 male and 12 female healthy volunteers underwent the Trier social stress test for groups (TSST-G). Heart rate variability was quantified by measuring the standard deviation (SDNN) and root mean square of successive differences (RMSSD) between normal-to-normal inter-beat intervals. Salivary α-amylase (sAA) activity was assessed as a surrogate for noradrenaline plasma concentrations. Oxidative DNA damage was determined using whole-blood single-cell gel electrophoresis ("tail moment" in the "comet assay"). A total of 33 subjects presented with a prospective risk of myocardial infarction (risk+) vs. 59 subjects without risk (risk-). The TSST-G stress significantly increased blood pressure, heart rate, and sAA in both groups, while oxidative DNA damage was only increased in the risk+ group. Immediately after the TSST-G, the "tail moment" showed significant inverse linear relations with both SDNN and RMSSD. Acute psychosocial stress may cause oxidative DNA damage, the degree of which is directly related to the individual cardiovascular risk profile and depends on the stress-induced increase in the sympathetic tone.

Differential quantitative analysis of MHC ligands by mass spectrometry using stable isotope labeling.

Currently, no method allows direct and quantitative comparison of MHC-presented peptides in pairs of samples, such as transfected and untransfected, tumorous and normal or infected and uninfected tissues or cell lines. Here we introduce two approaches that use isotopically labeled reagents to quantify by mass spectrometry the ratio of peptides from each source. The first method involves acetylation and is both fast and simple. However, higher peptide recoveries and a finer sensitivity are achieved by the second method, which combines guanidination and nicotinylation, because the charge state of peptides can be maintained. Using differential acetylation, we identified a beta catenin-derived peptide in solid colon carcinoma overpresented on human leucocyte antigen-A (HLA-A)(*)6801. Guanidination/nicotinylation was applied to keratin 18-transfected cells and resulted in the characterization of the peptide RLASYLDRV (HLA-A(*)0201), exclusively presented on the transfectant. Thus, we demonstrate methods that enable a pairwise quantitative comparison leading to the identification of overpresented MHC ligands.

Pro-inflammatory cytokines after different kinds of cardio-thoracic surgical procedures: is what we see what we know?

OBJECTIVE: Due to the combination of local trauma, extracorporeal circulation (ECC), and pulmonary and myocardial reperfusion, cardiac surgery leads to substantial changes in the immune system and possibly to post-operative complications. Procedures without ECC, however, have failed to demonstrate clear advantages. We hypothesized that ECC is far less important in this context than the reperfusion/reventilation of the lung parenchyma and the surgical trauma. We therefore conducted a prospective observational study to compare immune reactions after cardiac operations with those after thoracic surgery. METHODS: Serum levels of pro-inflammatory interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-alpha as well as C-reactive protein (CRP), lipoprotein-binding protein (LBP) and procalcitonin (PCT) were measured pre-operatively (d0), at the end of the operation (dx), 6h after the operation (dx+), on the 1st (d1), 3rd (d3), and 5th (d5) post-operative days in 108 patients (pts) undergoing elective coronary artery bypass grafting (CAB) with ECC (n=42, CPB CAB), off-pump coronary artery bypass surgery (n=24, OP CAB) without ECC or thoracic surgery (n=42, TS). RESULTS: After cardiac surgery (CS), IL-6 and IL-8 increased and reached a maximum on dx+. IL-6 returned to baseline values at d3, whereas IL-8 remained elevated until d5. No difference was found between OP CAB and CPB CAB patients. In the TS patients, IL-6 increased later (dx+) and absolute levels were lower than in the CS patients. No increase in IL-8 was noted in the TS patients. Due to the high variation in the results obtained in all three groups, there was no significant change in TNF-alpha. A comparison of TS, OP CAB, and CPB CAB revealed that the CS patients had higher levels on d0, dx, d3, and d5. Serum levels of CRP, LBP, and IL-2R increased from dx+ to d5 in all groups and reached maximum values on d3. Whereas we found no difference in CRP and IL-2R between the groups, LBP levels were significantly higher from dx+ to d3 after OP CAB. PCT was elevated from dx+ to d3 in all pts. Similar levels were noted for the TS and OP CAB patients. The CPB CAB patients showed the highest levels. CONCLUSIONS: Surgical trauma and reperfusion injury appear to represent the predominant factors resulting in immunologic changes after cardiac surgery. Cardiopulmonary bypass (CPB) may be less important for immune response and acute-phase reactions than previously suspected. In addition, our data indicate a relationship between IL-6 synthesis and the degree of surgical trauma. IL-8 appears to be elevated only after cardiac surgery whereas PCT liberation depended on the use of ECC.

Postoperative colonic motility increases after early food intake in patients undergoing colorectal surgery.

BACKGROUND: Stimulation of colonic motility by the gastrocolonic response may help to reduce inhibition of gastrointestinal motility after colorectal surgery. We aimed to investigate whether postoperative colonic motility is increased after early food intake. METHODS: Nineteen patients undergoing colorectal surgery and 7 healthy volunteers were investigated. Colonic motility was recorded with a combined manometry/barostat system, and the effect of a standard 500-kcal meal was evaluated once in healthy volunteers and in 15 patients on the first and second postoperative day. Four patients remained unfed, serving as controls. RESULTS: In patients, the colonic motility index increased from 12 +/- 5 at baseline to 65 +/- 24 mm Hg after the meal on postoperative day 1 (mean +/- SEM; P < .01), while barostat bag volumes decreased, indicating a rise in colonic tone. On day 2, the motility index was 62 +/- 17 mm Hg at baseline and did not change after the meal. In unfed controls, no change was observed during colonic motility recordings on both postoperative days. In healthy volunteers, the colonic motility index increased from 98 +/- 52 at baseline to 151 +/- 58 mm Hg postprandially (P < .05). CONCLUSIONS: As in healthy volunteers, there is a potential to stimulate colonic motility by early food intake in postoperative patients. This may help to improve prolonged colonic motility disorders after colorectal surgery.

Documentation of 7051 chronic wounds using a new computerized system within a network of wound care centers.

HYPOTHESIS: Wound care can be prospectively recorded on a large scale if a standardized wound documentation system is established. Based on such a documentation system, a network of specialized wound care centers can generate a large and valid database of wound characteristics, wound-healing dynamics, and wound care. DESIGN: A clinical prospective analysis. SETTING: Ten German specialized surgical wound care centers. PATIENTS AND METHODS: In a 2-year pilot phase, 4175 patients with 7051 chronic nonhealing wounds were documented using a new computerized system and treated following defined standards. RESULTS: A total of 1761 diabetic, 1349 venous, 1146 ischemic, 1079 pressure, and 759 postoperative nonhealing wounds and 957 ulcers with other causes were prospectively documented. Chronicity of ulceration was shown by the long wound duration of 433 days (range, 14-1867 days). Wound documentation was well integrated into daily practice, as shown by a mean +/- SD documentation time of 5.7 +/- 2.2 minutes per visit. A multivariate analysis of factors known to interfere with wound healing revealed significant effects of patient compliance, grading of wound depth, and patient age (P<.001 for all). CONCLUSIONS: The German Wound Net achieved, for the first time, centralized and prospective documentation of more than 7000 chronic wounds treated according to defined guidelines. This new concept of a network of specialized wound care centers working with standardized treatment plans and prospective documentation of chronic wounds may open a new dimension for wound-healing studies and may represent an optimal platform for multicenter trials.

Proinflammatory and antiinflammatory cytokines after cardiac operation: different cellular sources at different times.

BACKGROUND: Cardiac operation produces substantial alterations within the immune system, which possibly predispose postoperative complications. However, the interplay between proinflammatory and antiinflammatory reactions and the cells involved in this process are not completely clear. Therefore, we investigated serum levels, as well as synthesis patterns, of proinflammatory and antiinflammatory cytokines. METHODS: Serum levels and production of interleukin (IL) IL-5, IL-6, IL-10, tumor necrosis factor-alpha, and interferon-gamma, using a mixed cell culture, (ie, monocytes, macrophages, and lymphocytes), as well as a purified lymphocyte culture were measured preoperatively (day 0), on postoperative day 1, on postoperative day 3, and on postoperative day 5 in 25 patients undergoing cardiac operations and were compared with 10 healthy volunteers. RESULTS: Serum level and mixed cell culture, production of IL-6, tumor necrosis factor-alpha, and IL-10 increased on postoperative day 1, but decreased in lymphocyte culture. Base line values were reached on postoperative day 5. Interferon-gamma serum levels remained unchanged, whereas IL-5 serum levels increased on postoperative days 3 and 5. Cell culture synthesis showed a significant suppression for both mediators in both cell cultures, which returned to baseline on postoperative day 3 in mixed cell culture. Interferon-gamma production by lymphocytes was suppressed until postoperative day 5, whereas IL-5 returned to preoperative values on postoperative day 5. CONCLUSIONS: Cardiac operation induces a biphasic immune response. The first phase (postoperative day 1) appears to represent the proinflammatory and antiinflammatory reaction of the innate immune system returning to base line on postoperative day 3. The second phase (postoperative day 5) may represent the response of the adaptive immune system and is characterized by an antiinflammatory type of reaction. This may explain why the systemic inflammatory response occurs immediately after cardiac operation, whereas infections occur later.

Postoperative colonic motility in patients following laparoscopic-assisted and open sigmoid colectomy.

Clinical reports on laparoscopic-assisted sigmoid colectomy (LASC) suggest that the period of postoperative inhibition of gastrointestinal motility is shortened as compared to open sigmoid colectomy (OSC). We aimed to specifically investigate whether colonic motility increases more rapidly following LASC compared to OSC. LASC was performed in 11 patients and OSC in nine patients for recurrent diverticulitis or carcinoma. During surgery a manometry catheter was inserted into the colon via the anus, and the tip was placed in the splenic flexure. Continuous manometric recordings were performed from the day of surgery until postoperative day 3 with a four-channel microtransducer manometry system combined with a portable data logger. The postoperative colonic motility index was 101+/-18, 199+/-30, and 163+/-27 mm Hg/min on days 1, 2, and 3 after LASC, respectively, which was increased compared to indexes of 53+/-15, 71+/-18, and 76+/-23 following OSC (mean+/-standard error of the mean; P<0.05). The amplitude but not the frequency of contractions was higher following LASC compared to OSC. Following LASC, patients requested a similar amount of pain medication but resumed oral food more rapidly on postoperative days 2 and 3 (P<0.05), and they were discharged from the hospital earlier (P<0.05). Colonic motility in particular and the patient's condition in general seem to improve more rapidly following LASC compared to the open procedure.

Human duodenal spheroids for noninvasive intracellular pH measurement and quantification of regulation mechanisms under physiological conditions.

Three-dimensional cell cultures (spheroids) of biopsies of human duodenum were used to develop a new noninvasive method for studying intercellular and intracellular mechanisms. Through examinations of intracellular pH regulation, high functional similarity to native tissue could be shown, as already evidenced morphologically. A special microperfusion chamber was developed to fix individual spheroids physically to a nylon net, via laminar perfusion flow through the chamber. A significant improvement over current fixation methods was shown by the increase of cell viability almost up to 100%. Viability of the spheroids was confirmed by trypan blue exclusion, by a LIVE/DEAD viability/cytotoxicity kit, and by BCECF distribution. Intracellular pH was measured by use of the pH-sensitive fluorescence dye BCECF. To investigate the intracellular pH regulation, spheroid-like vesicles were acidified by NH4Cl prepulse technique. The subsequent active intracellular pH recovery was blocked with Na+-free Krebs Henseleit (KH) solution, with amiloride KH (inhibitor of the Na+-H+-exchanger), or with H2DIDS KH (inhibitor of the HCO3(-)-Cl(-)-exchanger and Na+-HCO3(-)-cotransporter). The intracellular pH of the spheroids was 7.31 +/- 0.05. pH-backregulation after acidification was prevented by sodium-free buffer, amiloride, and H2DIDS. These experiments indicated the presence of a Na+-H+-exchanger and a Na+-HCO3(-)-cotransporter. In conclusion, the human duodenal spheroid is an excellent physiological system for in vitro studies of the human duodenum.

Models to study ischemia in chronic wounds.

Wound healing is a complex immune response designed to achieve tissue repair following injury. Imbalance of stimulating and inhibiting factors cause failure of healing. Ischemia is a major cause of wound repair dysregulation and may be limb and life threatening. Investigating ischemic wound healing using animal models minimizes the complex accompanying factors that are usually present in humans, such as age or diabetes. This paper presents a limited review on normal physiological healing and on models that are used to study compromised healing under ischemic conditions.

Skin grafting of venous ulcers: a review of its current role.

As a therapeutic option, grafting of venous ulcers has not been very successfully received despite the different types of grafting methods. Currently, there are only a few controlled randomized trials offering clear guidance to clinicians. The development of artificially bioengineered skin constructs has led to a renewed interest in wound bed preparation, and preliminary successes suggest that the role of skin grafting could be studied in the current context.

Isotopic fingerprinting for the authenticity control of crop protection active compounds using the representative insecticide Fipronil.

Isotopic fingerprinting was evaluated for its potential to generate characteristic fingerprints of crop protection products in an extensive survey, using the insecticide Fipronil. One hundred and twenty batches of Fipronil from the BASF production site in France were analyzed for the isotope ratios of δ(13)C, δ(15)N, and δ(34)S. Samples spanned a production time of four years and were analyzed by elemental analysis, coupled to isotope ratio mass spectrometry (EA/IRMS). A number of Fipronil samples from other sources were analyzed in the same manner and were compared to the samples from BASF by means of multivariate data analysis. The isotopic fingerprint was sufficiently specific to differentiate between Fipronil from BASF production and Fipronil from other producers. This suggests that isotopic fingerprinting is suitable for the authenticity control of active compounds in crop protection products. It is anticipated that this technique will deliver great benefit in the defense against counterfeits and illegal parallel imports.

The challenge of military surgical education.

Now that field hospitals and rescue stations have been provided with state-of-the-art equipment, it is important to tailor the medical qualifications of military surgeons to the specific requirements of missions outside Germany. The objective of this article is to provide guidelines for a new training model. Einsatzchirurgie is defined as surgical treatment provided under restricted conditions in an unfamiliar environment. Its purpose is, first and foremost, to provide emergency treatment. The spectrum of Einsatzchirurgie, however, also encompasses maximum medical treatment on a case-by-case basis and emergency surgical treatment in a mass casualty situation. Training, for example, may consist of a 6-year basic training course in surgery followed by specialist training in abdominal surgery, traumatology, and courses in further disciplines--e.g., urology, gynecology, or neurosurgery. In addition to the qualifications required by the Landesärztekammem (professional organizations of German physicians at the federal countries level), military surgeons who are to become senior medical officers should also be qualified to provide immediate non-surgical emergency care. In these times, the education of military surgeons remains a great challenge. Motivation to work as a surgeon in the armed forces with multiple deployments during the career requires enthusiasm and professionalism. The attractiveness of the training institution and the home hospital is key for successful work as a military surgeon.

CD8+ T-cell response against MUC1-derived peptides in gastrointestinal cancer survivors.

The tumor-associated antigens CEA, MUC1 and Her2/neu are broadly expressed in gastrointestinal tumors, and are attractive candidates for targeting by T-cell-based immunotherapy. However, little is known about the natural cytotoxic T-cell response of patients suffering from colorectal or gastric carcinoma against these three as well as other antigens. Using a quantitative reverse transcription-polymerase chain reaction-based assay for IFN-gamma, we analyzed the CD8+ T-cell repertoire present in the blood of HLA-A2+ gastrointestinal tumor survivors against five known epitopes derived from CEA, MUC1 and Her2/neu. The results show that most of the patients (16 from 22 tested) have detectable, peripheral CD8+ T cells directed against at least one of these three proteins. Interestingly, the majority of these patients reacts to the two MUC1-derived HLA-A*0201 epitopes tested (14 from 16), demonstrating that this protein represents one dominant target for CD8+ T cells in gastrointestinal cancer.

Functional asymmetry of pelvic floor innervation and its role in the pathogenesis of fecal incontinence.

UNLABELLED: While the regular and symmetric innervation of the pelvic floor has been regarded as "established" for many years, recent data indicate that asymmetry of innervation of the sphincters may exists and may contribute to the occurrence and severity of incontinence symptoms in case of pelvic floor trauma. METHODS: A systematic review of published papers on asymmetry of sphincter innervation was performed including studies in healthy volunteers and patients with incontinence. 234 consecutive patients with fecal incontinence were investigated by means of side-separated mass surface EMG from the left and right side anal canal, these data were correlated to clinical and anamnestic findings. RESULTS: The literature survey indicates that asymmetry of sphincter innervation exists in a subgroup of healthy male and female volunteers, and may be a risk factor to become incontinent in case of trauma. Patients with incontinence in whom asymmetry of sphincter innervation could be shown more frequently reported a history of pelvic floor trauma during childbirth. Childbirth per se but not the number of deliveries predicted sphincter asymmetry. Asymmetrically innervated sphincters show a compromised sphincter function in routine anorectal manometry. CONCLUSION: Assessment of sphincter innervation asymmetry may be of value in clinical routine testing of patients with incontinence. However, a new technology is needed to replace mass surface EMG by multi-electrode arrays on a sphincter probe. This is one of the goals of the EU-sponsored research project OASIS.

Xanthogranulomatous cholecystitis resembling carcinoma with extensive tumorous infiltration of the liver and colon.

BACKGROUND: Xanthogranulomatous cholecystitis is a macrophage-rich inflammatory condition of the gallbladder that occasionally presents with tumorlike appearance. CASE PRESENTATION: In the present case the inflammation involved all the layers of the gallbladder, the surrounding connective tissue, and part of the right lobe of the liver and right transverse colon. The clinical and radiological findings were suggestive of advanced carcinoma of the gallbladder. However, intraoperative frozen section investigation revealed xanthogranulomatous cholecystitis, for which simple cholecystectomy is the treatment of choice. CONCLUSIONS: The original cause of the condition is unclear in most cases. In the present case it is possible that rupture of the gallbladder in association with the patient's known history of trauma have initiated the process.